Triose phosphate isomerase (TIM) is one of the fastest enzymes known. Its great speed and efficiency have made it the subject of much research designed to elucidate its catalytic mechanism. Kinetic and labeling studies have derived the free energy diagram of the reaction and have shown the presence of an enzyme bound intermediate. Spectroscopic and NMR techniques have demonstrated the properties of certain active site residues and of the bound substrate. In our lab, yeast TIM has been crystallized and the structure determined to atomic resolution. As a result, we and others have located the active site residues and have suggested a likely chemical mechanism for the reaction. Unfortunately, there are few precise methods for the accurate testing of theories on the specific function of the active site residues. However, we are in the position to use the most precise of the current methods, directed mutagenesis of codons for the active site residues. In collaboration with others, we have cloned and sequenced the gene for yeast triose phosphate isomerase (yTPI). This gene is now in a form suitable for modification by the method of primer directed mutagenesis, which is capable of introducing single base changes in the protein coding sequence. With this approach, we hope to produce TIM proteins with altered active site structures whose properties will test our theories of catalysis. Investigations of the properties of the mutant enzymes will be made by both biochemical and crystallographic methods.